N-myristoyl transferase (NMT) is a monomeric enzyme, which is ubiquitous in eukaryotes. NMT catalyses an irreversible co-translational transfer of myristic acid (a saturated 14-carbon fatty acid) from myristoyl-Coenzyme A (myr-CoA) to a protein substrate containing an N-terminal glycine with formation of an amide bond (Farazi, T. A., G. Waksman, and J. I. Gordon, J. Biol. Chem., 2001. 276(43): p. 39501-39504). N-myristoylation by NMT follows an ordered Bi-Bi mechanism. Myr-CoA binds to NMT in the first NMT binding pocket prior to the binding of a protein substrate (Rudnick, D. A., C. A. McWherter, W. J. Rocque, et al., J. Biol. Chem., 1991. 266(15): p. 9732-9739.). The bound myr-CoA facilitates the opening of a second binding pocket where the protein substrate binds. Following binding of the protein substrate, transfer of myristate to the protein substrate takes place via a nucleophilic addition-elimination reaction, finally with the release of CoA and the myristoylated protein.
NMT plays a key role in protein trafficking, mediation of protein-protein interactions, stabilization of protein structures and signal transduction in living systems. Inhibition of the NMT enzyme has the potential to disrupt multi-protein pathways, which is an attractive characteristic to reduce the risk of the development of resistance in, for example, treatment or prophylaxis of microbial infections and hyperproliferative disorders.
Biochemical analysis has shown high conservation of myr-CoA binding sites, but divergent peptide binding specificities between human and fungal and parasitic NMTs (Johnson, D. R., R. S. Bhatnagar, J. I. Gordon, et al., Annu. Rev. Biochem., 1994. 63: p. 869-914.). As a consequence, NMT can be viewed as a target with the potential for the development of selective non-peptidic inhibitors.
NMT fungal and mammalian enzymes from various sources have been well characterized, see for example the following references: Saccharomyces cerevisiae (Duronio, R. J., D. A. Towler, R. O. Heuckeroth, et al., Science, 1989. 243(4892): p. 796-800), Candida albicans (Wiegand, R. C., C. Carr, J. C. Minnerly, et al., J. Biol. Chem., 1992. 267(12): p. 8591-8598) and Cryptococcus neoformans (Lodge, J. K., R. L. Johnson, R. A. Weinberg, et al., J. Biol. Chem., 1994. 269(4): p. 2996-3009), human NMT1 (McIlhinney, R. A. J. and K. McGlone, Exp. Cell Res., 1996. 223: p. 348-356) and human NMT2 (Giang, D. K. and B. F. Cravatt, J. Biol. Chem., 1998. 273: p. 6595-6598).
NMT has also been characterised in protozoan parasites. See for example the following references: Plasmodium falciparum (Pf) (Gunaratne, R. S., M. Sajid, I. T. Ling, et al., Biochem. J., 2000. 348: p. 459-463), Plasmodium vivax (Pv), Leishmania major (Lm) (Price, H. P., M. R. Menon, C. Panethymitaki, et al., J. Biol. Chem., 2003. 278(9): p. 7206-7214.), Leishmania donovani (Ld) (Branningan, J. A., B. A. Smith, Z. Yu, et al., J. Mol. Biol., 2010. 396: p. 985-999) and Trypanosoma brucei (Tb) (Price, H. P., M. R. Menon, C. Panethymitaki, et al., J. Biol. Chem., 2003. 278(9): p. 7206-7214.
Several myristoylated proteins have been observed in protozoans and their functions have been determined. These proteins and the processes in which they are involved suggest that N-myristoylation may play a role in multiple pathways in the biology of parasites. Inhibition of myristoylation could thus disrupt multiple pathways. The potential for the development of resistance should thus be smaller than for some other targets. To date, only a single isoform of NMT has been found in each protozoan organism investigated. If it is correct that there is only a single isoform, then that will also assist in reducing the potential for the development of resistance.
Inhibition of human NMT has also been suggested as a target for treating or preventing various diseases or disorders, for example hyperproliferative disorders (cancers, e.g. human colorectal cancer, gallbladder carcinoma, brain tumors, lymphomas such as B-cell lymphoma) (Resh M D. 1993. Biochern. Biophys. Acta 1115, 307-22), and viral infections such as HIV (Gottlinger H G, Sodroski J G, Haseltine W A. 1989. Proc. Nat. Acad. Sci. USA 86:5781-85; Bryant M L, Ratner L. 1990. Proc. Natl. Acad. Sci. USA 87:523-27) and human rhinovirus (HRV) (Davis M P, Bottley, G, Beales L P, Killington, R A, Rowlands D J, Tuthill, T J, 2008 Journal of Virology 82 4169-4174).
As described above, there are two binding pockets in NMT. One is the myr-CoA binding pocket and the other is the peptide binding pocket. Most NMT inhibitors reported to date target the peptide binding pocket. Most NMT inhibitors developed to date have been targeted to fungal N-myristoyl transferases.
Compounds active as inhibitors of NMT have previously been disclosed, see for example WO00/37464 (Roche), WO2010/026365 (University of Dundee), and WO2013/083991 (Imperial Innovations Limited). In addition, Bell et al disclosed the results of a high throughput screening study carried out to identify inhibitors of NMT, and disclosed the compound N,N-dimethyl-1-(5-(o-tolyl)-1H-indazol-3-yl)methanamine as having activity against Plasmodium falciparum NMT (PLoS Neglected Tropical Diseases, 2012, 6, e1625). A further indazole-containing analogue (1-(5-(4-fluoro-2-methylphenyl)-1H-indazol-3-yl)-N,N-dimethylmethanamine) was disclosed as part of a presentation “Selective inhibitors of protozoan protein N-myristoyl transferases” on 18 Sep. 2012 during a Symposium “Emerging Paradigms in Anti-Infective Drug Design” held at the London School of Hygiene and Tropical Medicine.
However, there remains a need in the art for further compounds active as inhibitors of N-myristoyl transferase.